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首页> 外文会议>Scale-up and manufacturing of cell-based therapies V >DEVELOPMENT OF A SCALE-DOWN APPROACH TO THE SCALABLE CULTURE OF INDUCED PLURIPOTENT STEM CELLS ON MICROCARRIERS USING SINGLE-USE VERTICAL-WHEEL~(TM) BIOREACTORS UNDER XENO-FREE CONDITIONS
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DEVELOPMENT OF A SCALE-DOWN APPROACH TO THE SCALABLE CULTURE OF INDUCED PLURIPOTENT STEM CELLS ON MICROCARRIERS USING SINGLE-USE VERTICAL-WHEEL~(TM) BIOREACTORS UNDER XENO-FREE CONDITIONS

机译：在无氙气条件下使用单次使用Vertical-Wheel〜（TM）生物反应器的微型培养物中可诱导的多能干细胞可缩放培养的按比例缩小方法的开发

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Induced Pluripotent Stem Cells (iPSC) are capable of extensive self-renewal while retaining the ability to differentiate into virtually all cell types of the body. These cells are the subject of much research and development activity aimed at the development of cell-based tools, which may speed drug discovery, and cell-based medical therapies that are being developed to address unmet medical needs. However, development of these therapies is hampered by manufacturing bottlenecks including production scale up to meet the anticipated demand. PBS Biotech, Inc. has developed a single use bioreactor with an innovative Vertical-Wheel™ design that promotes more homogenous and gentle particle suspension, under lower hydrodynamic shear environment than traditional bioreactor vessel design. Vertical-Wheel bioreactors are available from lab-scale vessels (PBS MINI) to larger production units (up to 500L). This study describes the culture of human iPSCs on microcarriers under xeno-free conditions using Vertical-Wheel bioreactors. Human iPSCs were cultured on microcarriers to provide surface for cell attachment using the chemically defined Essential 8 culture medium, a xeno-free, feeder-free culture medium. The culture conditions were optimized in terms of 1) initial cell/microcarrier ratio, 2) inoculation method and 3) agitation rate, in the PBS-0.1 vessel using 80 mL working volume. The cells were successfully expanded, up to a 7-fold increase in cell number, after 6 days in the bioreactor. Glucose consumption and lactate production were analyzed to prevent glucose starvation or excessive lactate accumulation. These optimized culture conditions were successfully repeated in a larger vessel, the PBS-0.5 using 300 mL working volume, demonstrating the scalability of the Vertical-Wheel system. With this PBS-0.5 bioreactor, 3 x 10~8 cells were produced after 6 days of operation, and the specific growth rate (0.72 day~(-1)) was similar to the one observed with the PBS-0.1 (0.68 day~(-1)). The applications of iPSC cells and their progeny, especially in clinical settings, will require a guarantee of cell quality. After PBS-MINI bioreactor culture, the expression of pluripotency markers, such as Oct4, Nanog, and SSEA4 was assessed by immunocytochemistry and flow cytometry. The directed differentiation into the neural lineage of the expanded cells was performed and the pluripotency of the cells was further tested after embryoid body formation. The robustness of this process method was evaluated by cultivating another iPSC cell line under the same process conditions, resulting in identical growth kinetics in the PBS MINI-0.1. The methodology developed herein, which grows human iPSC on microcarriers in single-use bioreactors using chemically defined xeno-free cultivation reagents provides a foundation upon which further refinement and scale-up of processes can be built for large scale production of iPSCs.
机译：诱导多能干细胞（iPSC）能够进行广泛的自我更新，同时保留分化为几乎所有人体细胞类型的能力。这些细胞是许多旨在开发基于细胞的工具（可能会加快药物开发速度）和正在开发的基于细胞的药物治疗方法的主题，这些工具正在开发中以解决未满足的医疗需求。然而，这些疗法的发展受到制造瓶颈的阻碍，包括生产规模扩大以满足预期需求。 PBS Biotech，Inc.开发了一种具有创新性Vertical-Wheel™设计的一次性生物反应器，该设计在比传统生物反应器容器设计更低的流体动力剪切环境下，可促进更均匀且温和的颗粒悬浮。垂直轮生物反应器可用于实验室规模的容器（PBS MINI）到较大的生产装置（最大500L）。这项研究描述了使用立式轮式生物反应器在无异种条件下在微载体上培养人iPSC。使用化学成分定义的Essential 8培养基（无异种，无饲养层的培养基）在微载体上培养人iPSC，以提供细胞附着的表面。在1）初始细胞/微载体比例，2）接种方法和3）搅拌速度方面，在PBS-0.1容器中使用80 mL工作体积对培养条件进行了优化。在生物反应器中放置6天后，细胞成功扩增，细胞数量最多增加了7倍。分析了葡萄糖的消耗和乳酸的产生，以防止葡萄糖饥饿或过多的乳酸积累。这些优化的培养条件已在使用300 mL工作体积的较大容器PBS-0.5中成功重复，证明了Vertical-Wheel系统的可扩展性。使用该PBS-0.5生物反应器，在运行6天后可产生3 x 10〜8个细胞，比生长速率（0.72天〜（-1））与使用PBS-0.1观察到的比生长速率（0.68天〜 （-1））。 iPSC细胞及其后代的应用，尤其是在临床环境中，将需要保证细胞质量。 PBS-MINI生物反应器培养后，通过免疫细胞化学和流式细胞仪评估多能性标志物，例如Oct4，Nanog和SSEA4的表达。进行定向分化为扩增细胞的神经谱系，并在形成拟胚体后进一步测试细胞的多能性。通过在相同的工艺条件下培养另一种iPSC细胞系来评估该方法的耐用性，从而在PBS MINI-0.1中获得相同的生长动力学。本文开发的方法使人iPSC在一次性生物反应器中的微载体上生长，该方法使用化学成分明确的无异种培养试剂，为进一步构建和扩大工艺规模以大规模生产iPSC提供了基础。

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